We have recently identified five different cell types in the mammalian kidney that can form prostaglandins (PGs). We suspect the reason so many agents and treatments (e.g., hormones, nerve stimulation, ischemia) cause renal PG synthesis is that selective stimulation of PG formation occurs at different cellular sites under various experimental conditions. In order to clarify the functional roles that PGs perform in renal physiology, it will be important to define both the parameters regulating the synthesis of and the nature of the PG derivatives produced by each of the PG-forming cells. Those segments of the nephron itself where PGs are produced are cells which interact with antidiuretic hormone(ADH)--namely epithelial cells of the cortical and medullary collecting tubules. We have devised procedures for isolating rabbit collecting tubule cells which are responsive to ADH and have labeled the cellular lipids with radioactive fatty acids. We now propose to investigate PG biosynthesis by collecting tubule cells and in particular, to determine the potential relationships between ADH binding, 3',5'-cAMP formation and PG biosynthesis. Initially, we will characterize the PG products formed by these cells both from synthetic 3H-PG endoperoxides and from endogenous radiolabeled lipids. Second, we will determine which hormonal agents cause selective release of arachidonic acid from cells prelabeled with 14C-linoleic and 3H-arachidonic acids and will identify the esterified lipid precursor. We will determine if intracellular 3',5'-cAMP levels influence the release of arachidonate or its subsequent oxygenation. In addition, we plan to pursue ongoing studies on the subcellular location of the cyclooxygenase which catalyzes formation of PG endoperoxides both in bovine kidney collecting tubules and vascular endothelial cells using immunohistochemical labeling of the enzyme. Our results should provide insights into how PGs formed by the kidney modulate natriuresis and renal blood flow.